A family of cold-regulated RNA-binding protein genes in the cyanobacterium Anabaena variabilis M3.

I previously found a cold-regulated RNA-binding protein gene rbpA (now named rbpA1) in Anabaena variabilis M3 [Sato, N. (1994) Plant Mol. Biol. 24, 819-823]. I show here that this gene is a member of a gene family containing at least eight members as evidenced by Southern blot and immunoblot analyses. I have isolated three additional genes (rbpB, rbpC and rbpD) in this family. Of these, rbpB was 100% identical to the rbpB gene of Anabaena 7120 reported previously. Another gene named rbpA in Anabaena 7120 was also found to exist in A.variabilis M3 with identical sequence and named rbpA2. The amino acid sequences of these gene products were highly conserved, except that the RbpD protein lacked glycine-rich C-terminal domain present in all other known members of the gene family. RNA blot and immunoblot analyses showed that the expression of rbpA1, rbpA2, rbpB, rbpC and rbpD, as well as uncloned rbp genes was regulated by cold, though the exact time-course and extent of response to cold were different among these genes. Gel-filtration assay showed that all of the Rbp proteins have higher affinities to poly(G) and poly(U) than to poly(A) and poly(C).     

Ammonia-excreting mutant strain of the cyanobacterium Anabaena variabilis supports growth of wheat

Summary Growth of wheat in a nitrogen-free hydroponic co-culture with a mutant strain of the cyanobacterium Anabaena variabilitis (strain SA-1) was enhanced over plants grown with the parent strain SA-0. This increase was achieved in the dry weight, grain yield, and total nitrogen content of the plants. Nitrogenase activity of the mutant strain SA-1 was increased in a co-culture of the cyanobacterial mutant with wheat plants compared to the activity of the wild-type strain in association with wheat. Offprint requests to: M. Gunasekaran     

Photophobic responses of the cyanobacterium Anabaena variabilis

The photophobic responses in the Cyanobacterium Anabaena variabilis which belongs to the Nostocaceae have been studied with aid of a population method as well as by single trichome observations. In white light experiments both step-up and step-down photophobic responses were observed. The wavelength dependence was examined at a constant fluence rate. The photophobically active light is absorbed by the photosynthetic pigments, mainly by the phycobiliproteins and chlorohyll a. Above 690 nm only negative reactions were observed, i.e. the trichomes left the light trap. In white light experiments DCMU strongly inhibited the photophobic responses, whereas photokinesis was not affected to the same extent indicating that the reaction is coupled with the non cyclic photosynthetic electron transport. DBMIB impaired the photophobic behaviour only slightly. It seems that the photophobic responses of A. variabilis are controlled by a similar mechanism as in Phormidium uncinatum (Oscillatoriaceae) although the two families and, hence, the two species differ in their movement mechanism as well as in their photoactic behaviour.     

Transport of leucine in the cyanobacterium Anabaena variabilis

The amino acid leucine was transported by the cyanobacterium Anabaena variabilis. The K _m for transport was 10.8 M; the V _max was 8.7 nmoles min^–1 mg^–1 chlorophyll a. Transport of leucine was energy dependent: uptake of leucine was inhibited in the dark, and by DCMU and cyanide. Transport was neither dependent on nor enhanced by Na⁺. Prior growth of cells with leucine did not repress transport of [¹⁴C]-leucine. Alanine, glycine, valine, and methionine were strong competitive inhibitors of leucine uptake; serine, threonine, isoleucine, norleucine, and d-alanine competitively inhibited to a lesser degree. Other amino acids or amino acid analogues, including d-leucine, -aminoisobutyrate, and d-serine did not inhibit the transport of leucine.Abbreviations Chl a chlorophyll a - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - TES N-tris(hydroxymethyl)-2-aminoethane-sulfonic acid - TCA trichloroacetic acid - Tris N-tris(hydroxymethyl)aminoethane     

New low-copy plasmid in cyanobacterium Anabaena variabilis

Complete genome sequencing was performed for Anabaena variabilis ATCC 29413 from the collection of the Chair of Genetics, Department of Biology, Moscow State University, Russia. In addition to known plasmids A, B, and C, a new circular low-copy plasmid was detected and named D. It was also sequenced completely and found to have 27051 bp. The plasmid contained the parA and parB genes of the partition system, two genes that encode replication proteins, a gene for site-specific recombinase, a type-I restriction-modification system, and several genes with unknown functions. Analysis by PCR revealed the presence of plasmid D in two epiphytic strains from Vietnam, i.e., Anabaena sp. 182 and Anabaena sp. 281, as well as in Anabaena sp. V5 and A. azollae (Newton’s isolate).     

Induction of nitrate assimilation in the cyanobacterium Anabaena variabilis

Filaments of Anabaena variabilis Kütz strain ATCC 29413 grown in the absence of nitrate contain nitrate reductase that is active in permeabilized filaments, but not in intact, living filaments until they have been incubated for about 40 min in the presence of nitrate. The delayed acquisition of the ability to reduce nitrate is insensitive to chloramphenicol. Thus, switching on of enzyme activity in the presence of nitrate does not involve protein synthesis and nitrate reductase activity is not regulated by the amount of enzyme present.     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine     

Structural aspects of akinete germination in the cyanobacterium Anabaena variabilis

Electronmicroscopical investigations of light activated akinetes in different phases before outgrowth of the germinating cell showed two alterations in the akinete envelope, obviously in connection with the germination process. After induction of germination the akinetes show formation of an expanding more or less electron dense layer between the outer cell wall layer (outer membrane, L_IV) and the condensed part of the akinete coat (the transformed sheath of the vegetative cell). Between this new formed layer and the mentioned part of the akinete coat thick laminar layers are deposited which contain alternately electron dense and electron transparent strata. The expanding layer is assumed to be a mucous layer which acts as swelling body causing, after bursting of the layered shell, the expulsion of the germinating cell in the manner characteristic for Anabaena variabilis.     

Transport of molybdate in the cyanobacterium Anabaena variabilis ATCC 29413

Heterocyst-forming filamentous cyanobacteria, such as Anabaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase. A. variabilis efficiently transported (99)Mo (molybdate) at concentrations less than 10(-9) M. Competition experiments with other oxyanions suggested that the molybdate-transport system of A. variabilis also transported tungstate but not vanadate or sulfate. Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase. Transport of (99)Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate. Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase. These data provide evidence for a molybdate storage system in A. variabilis. NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate. The closely related strain Anabaena sp. PCC 7120 transported (99)Mo in a very similar way to A. variabilis.     

Photoinhibition and its wavelength dependence in the cyanobacterium Anabaena variabilis

In the cyanobacterium Anabaena variabilis the dependence of photoinhibition on fluence rate, duration and wavelength of irradiation were studied by measurements of oxygen production and fluorescence emission spectra. The analysis of the photosynthetic activity revealed that photoinhibition affects exclusively photosystem II (PS II), whereas photosystem I (PS I) remained largely unimpaired. Furthermore, PS II fluorescence emission decreased much faster in bleached than in unbleached controls.Studying the wavelength dependence of photoinhibition it was found that only radiation between 520 and 680 nm causes photoinhibition. This is about the same range of wavelengths which causes photobleaching. Fluorescence emission spectra of samples exposed to high fluence rates of 582 and 662 nm, respectively, essentially agree with those samples exposed to high fluence rates of white light, whereas the fluorescence emission spectra of samples exposed to blue light resemble those exposed to dim white light.NaN₃, a substance which prevents photobleaching, inhibits the photosynthetic O₂ production of Anabaena and, hence, enhances the photoinhibitory effect.     

Novel DNA-binding proteins in the cyanobacterium Anabaena sp. strain PCC 7120

As an approach towards elucidation of the biochemical regulation of the progression of heterocyst differentiation in Anabaena sp. strain PCC 7120, we have identified proteins that bind to a 150-bp sequence upstream from hepC, a gene that plays a role in the synthesis of heterocyst envelope polysaccharide. Such proteins were purified in four steps from extracts of vegetative cells of Anabaena sp. Two of these proteins (Abp1 and Abp2) are encoded by neighboring genes in the Anabaena sp. chromosome. The genes that encode the third (Abp3) and fourth (Abp4) proteins are situated at two other loci in that chromosome. Insertional mutagenesis of abp2 and abp3 blocked expression of hepC and hepA and prevented heterocyst maturation and aerobic fixation of N(2).     

Regulation of uridylic acid biosynthesis in the cyanobacterium Anabaena variabilis.

The pathway of uridylic acid biosynthesis established by Leiberman, Kornberg, and Simms has been shown to be operative in the filamentous cyanobacterium Anabaena variabilis. The only enzyme of uridylic acid biosynthesis found to be lacking in two uracil-requiring strains of A. variabilis was aspartate transcarbamylase, the first enzyme in the pathway of de novo biosynthesis of uridvlic acid. Neither uracil-limited growth of a uracil-requiring mutant nor growth of the wild type in high concentrations of uracil resulted in substantial changes in the specific activities of enzymes of uridylic acid biosynthesis. It is therefore concluded that A. variabilis does not regulate all enzymes of this pathway by means of repression. However, control of the flow of intermediates through this pathway is possible by feedback inhibition of aspartate transcarbamylase by a variety of nucleotides.     

Phosphate transport and arsenate resistance in the cyanobacterium Anabaena variabilis.

Cells of the cyanobacterium Anabaena variabilis starved for phosphate for 3 days took up phosphate at about 100 times the rate of unstarved cells. Kinetic data suggested that a new transport system had been induced by starvation for phosphate. The inducible phosphate transport system was quickly repressed by addition of Pi. Phosphate-starved cells were more sensitive to the toxic effects of arsenate than were unstarved cells, but phosphate could alleviate some of the toxicity. Arsenate was a noncompetitive inhibitor of phosphate transport; however, the apparent Ki values were high, particularly for phosphate-replete cells. Preincubation of phosphate-starved cells with arsenate caused subsequent inhibition of phosphate transport, suggesting that intracellular arsenate inhibited phosphate transport. This effect was not seen in phosphate-replete cells.     

High-affinity vanadate transport system in the cyanobacterium Anabaena variabilis ATCC 29413

High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 x 10(-9) M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri.